Listeria monocytogenes is a highly tractable intracellular pathogen and a cause of serious food-borne illness in humans. A primary determinant of L. monocytogenes pathogenesis is a secreted pore-forming protein referred to as listeriolysin O (LLO). LLO mediates escape of L.monocytogenes from a phagocytic vacuole and is absolutely essential for virulence. LLO has a number of unique properties that prevent its activity in the host cytosol including an acidic pH optimum and a PEST-like sequence. The goals of the current proposal are to identify both bacterial and host components that control LLO compartmentalization, and to determine why mutants that fail to properly compartmentalize LLO activity are avirulent. In Aim I, two genetic selection screens are proposed to identify bacterial proteins that control LLO secretion in a vacuole, and two other screens are proposed to identify bacterial proteins that prevent LLO secretion in the cytosol. The intracellular phenotypes of the mutants will be characterized in a variety of tissue culture assays, and secretion defects examined biochemically. The role of two recently identified autolysins whose synthesis or activation appears to be vacuole-specific will be examined. In Aim II, it will be determined why cytotoxic mutants are less virulent. One hypothesis that will be directly tested is that cytotoxic mutants become extracellular and are targeted by neutrophils. The role of neutrophils and neutrophil chemotaxis will be addressed in neutropenic and knockout mice. In Aim III, a functional genomics approach will be used to identify host proteins that control escape of L.monocytogenes from a vacuole and that prevent LLO toxicity in the host cytosol. The host cells to be used in these studies are a phagocytic Drosophila cell line that is extremely sensitive to double-stranded RNA interference (RNAi). RNAi specific for 7800 Drosophila genes will be produced and used to screen, microscopically, for host genes that encode proteins controlling LLO compartmentalization.